JP5672002B2 - Barley selection method - Google Patents

Description

  The present invention relates to a method for selecting barley, malt, and a malt fermented beverage.

  Malt extract is an important malt quality that is directly linked to economy in beer brewing. In addition, malt extract is a complex malt quality that involves various traits such as malt melt, enzyme strength, skin thinness, and crude protein content. Breeding barley with a high malt extract has become one of the major goals in barley breeding.

  In recent years, genome analysis has progressed even in barley, and a large amount of DNA marker information has become available. For this reason, in breeding institutions around the world, the quantitative trait loci (QTL) useful for breeding are detected by genetic analysis of malt quality, and there is an active movement to use them for breeding. Results have been reported (Non-Patent Documents 1 to 5).

  Regarding malt extract, the results of QTL analysis using various barley populations have been reported so far. In the Harrington × TR306 strain, chromosomes 1H and 5H (Non-patent Document 1), in the Harrington × Molex strain, chromosomes 1H and 2H (Non-patent Document 2), in the Chebec × Harlington strain, chromosome 5H (Non-patent Document 3), Galleon × Haruna Nijo QTLs related to malt extract are detected in chromosome 2H (Non-patent Document 4) in the strain, and chromosomes 1H, 2H, 5H, and 7H (Non-patent document 5) in the Steptoe × Molex strain.

D. E. Mother, et al, 1997, Crop Sci., 37, pp. 544-554 L. A. Marquez-Cedilo, et al, 2000, Theor. Appl. Genet. 101, pp. 173-184 A. R. Barr, et al, 2003, Australian J. Org. Agric. Res. , 54, pp. 1125-1130 H. M.M. Collins, et al, 2003, Australian J. Org. Agric. Res. , 54, pp. 1223-1240 P. M.M. Hayes, et al, 1993, Theor. Appl. Genet. 87, pp. 392-401

  In nonpatent literatures 1-5, the position on the chromosome from which QTL regarding a malt extract is detected changes with barley populations used. Possible causes are that parents do not have the gene, the number of strains used in the analysis, environmental factors, and the like. For this reason, the genetic region involved in the malt extract has not yet been clarified, and it is strongly desired to develop a DNA marker that is versatile for barley breeding.

  Then, an object of this invention is to provide the versatile barley selection method which can select barley with a high malt extract reliably.

  The present invention is a method for selecting barley having a high malt extract, wherein the test barley has a restriction enzyme HinfI recognition sequence (5′-GANTC-3) in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1. ') (In the recognition sequence, N represents an arbitrary base) A method for selecting the test barley as barley having a high malt extract is provided.

  In the present invention, the “MWG655A sequence” means a base sequence in the MWG655A locus. Further, “corresponds to the base sequence of SEQ ID NO: 1” means that it is at the same chromosomal location as the chromosomal location of the base sequence of SEQ ID NO: 1.

  In the barley selection method of the present invention, barley having a high malt extract is selected by determining whether or not the test barley has a recognition sequence for the restriction enzyme HinfI in the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1. can do. Since the barley selection method of the present invention is a selection method based on a genotype, barley having a high malt extract can be selected with high reliability without being affected by changes in the natural environment depending on the production area and year. Further, it is not necessary to collect a large amount of seed sample from the test barley, and barley having a high malt extract can be selected at the early stage of barley breeding (for example, the second generation of hybrids).

  Whether or not the test barley has the recognition sequence of the restriction enzyme HinfI in the MWG655A sequence is determined by using a primer set capable of amplifying the MWG655A sequence, and the polymerase chain reaction using the genomic DNA of the test barley as a template ( PCR) to obtain an amplified DNA fragment, and a step of digesting the amplified DNA fragment with the restriction enzyme HinfI. A DNA fragment cleaved by the restriction enzyme is obtained in the obtained restriction enzyme-treated product. This is preferably done by determining whether or not it exists.

  That is, the present invention is also a method for selecting barley with a high malt extract, using a primer set capable of amplifying the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1, and using the genomic DNA of the test barley as a template And a digestion step of digesting the amplified DNA fragment with the restriction enzyme HinfI. In the restriction enzyme-treated product obtained in the digestion step, Provided is a method for selecting the test barley as barley having a high malt extract when a cleaved DNA fragment is present.

  This barley selection method is based on the so-called Cleaved Amplified Polymorphic Sequence (CAPS) method in which a DNA fragment amplified by PCR is treated with a restriction enzyme HinfI to detect a polymorphism. As a result, the work becomes easier and it is possible to process multiple samples in a shorter time. Moreover, since selection with respect to many test barleys can be performed simultaneously, it is suitable for barley selection at a breeding site.

  The primer set is composed of a forward primer and a reverse primer, at least one of which is a base sequence of SEQ ID NO: 1 in the result of multiple alignment of the base sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Those that anneal to the polymorphic site in the nucleotide sequence of SEQ ID NO: 1 that is different from the sequence are preferred.

  The MWG655A locus has orthologs (MWG655B, MWG655C, and MWG655e loci) that have very high nucleotide sequence homology. By using such a primer set for PCR, the MWG655A locus is derived from the MWG655B and MWG655C loci. Nonspecific amplification of DNA fragments can be suppressed. Therefore, the selection efficiency can be improved, and the labor required for selection can be reduced.

  From the viewpoint of more surely suppressing non-specific amplification of the DNA fragment, the primer set is more preferably, for example, that the forward primer includes the 1st to 11th nucleotide sequences of the nucleotide sequence of SEQ ID NO: 1, Further, a forward primer consisting of the base sequence of SEQ ID NO: 4 and a reverse primer consisting of the base sequence of SEQ ID NO: 5 are particularly preferred.

  The present invention also provides a progeny line of barley that can be obtained by crossing barley selected by the above-mentioned method for selecting barley.

  The barley selected by the method of the present invention matches the genotype of the DNA marker in the MWG655A locus that affects the malt extract. And the barley of the progeny of the cross obtained by crossing these barleys has the same genotype as the parents with respect to the above DNA marker, so that it becomes barley with a high malt extract.

  The present invention also provides a malt production method including a malting process for producing barley selected by the above barley selection method or barley of the above-mentioned hybrid progeny line. Moreover, the malt which can be obtained by this method is provided.

  The malt of the present invention is obtained by malting barley selected by the above barley selection method or barley of the above-mentioned hybrid progeny line. Therefore, the malt extract is high and useful as a raw material for malt fermented beverages.

  The present invention is also a method for producing a malt fermented beverage comprising a preparation step and a fermentation step, wherein, in the preparation step, barley selected by the above-mentioned barley selection method, barley of the above-mentioned hybrid progeny line or the above-mentioned malt is used as a raw material Provide a way to do it. Moreover, the malt fermented drink which can be obtained by this method is provided.

  In the method for producing a malt fermented beverage of the present invention, in the preparation step, barley selected by the barley selection method, barley of the above-mentioned hybrid progeny line or the malt is used as a raw material, so that more malt from the same amount of raw material. A fermented drink can be manufactured and the manufacturing cost can be reduced. Moreover, since the malt extract contained in the raw material is high, a malt fermented beverage having a high nutrient content can be produced.

  ADVANTAGE OF THE INVENTION According to this invention, the versatile barley selection method which can select barley with high malt extract reliably can be provided. In addition, barley and malt having a high malt extract, and a high-quality malt fermented beverage made from such barley and malt are provided.

It is the multiple alignment result of the base sequence of sequence number 1, sequence number 2, and sequence number 3. It is a graph which shows the relationship between the genotype of the DNA marker used by the barley selection method of this invention, and a malt extract. It is an agarose gel electrophoresis image of the HinfI processed product of the PCR product obtained when the barley selection method of the present invention is carried out. It is a graph which shows the grade of the malt extract of barley selected by the method of this invention.

  Hereinafter, the present invention will be described in detail.

[Barley selection method]
The barley selection method of the present invention is a method for selecting barley having a high malt extract, and the test barley has a restriction enzyme HinfI recognition sequence (5 ′) in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1. -GANTC-3 ′) (in the recognition sequence, N represents an arbitrary base) This is a method of selecting the test barley as barley having a high malt extract.

  In the present invention, “malt extract” means the malt extract content per dry weight. The extract content rate of malt can be calculated | required from the extract content rate of a congress wort, for example. The extract content of the conger wort can be calculated from the increase in the specific gravity of the conger wort relative to the specific gravity of water. This increase in specific gravity is due to, for example, sugar, amino acids, vitamins and the like.

  In the present invention, the “MWG655A sequence” means a base sequence in the MWG655A locus. Further, “corresponds to the base sequence of SEQ ID NO: 1” means that it is at the same chromosomal location as the chromosomal location of the base sequence of SEQ ID NO: 1. The base sequence “corresponding to the base sequence of SEQ ID NO: 1” may have, for example, a mismatch of about several bases due to a single nucleotide polymorphism (SNP) with the base sequence of SEQ ID NO: 1.

  As a method for determining whether or not the test barley has a recognition sequence for the restriction enzyme HinfI in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1, a method widely used in this technical field may be employed. it can. Examples of such a method include a restriction fragment length polymorphism (RFLP) method, a sequencing method, and a CAPS method.

  The determination by the RFLP method can be performed as follows, for example. First, the genomic DNA of the test barley is digested with the restriction enzyme HinfI, the obtained DNA fragment (HinfI treated product) is subjected to gel electrophoresis, and further transferred to a membrane. Then, a probe capable of detecting the base sequence of SEQ ID NO: 1 is brought into contact with the membrane, and the resulting hybridization is detected. By analyzing the size and / or number of DNA fragments in which hybridization has been detected, it can be determined whether or not a recognition sequence for the restriction enzyme HinfI is present in the MWG655A sequence. By using genomic DNA of Mikamo golden species or Harrington species as a control, the determination can be made more easily.

  Examples of the probe capable of detecting the base sequence of SEQ ID NO: 1 include a probe having a sequence including the base sequence of SEQ ID NO: 1, a probe comprising the base sequence of SEQ ID NO: 1, and a part of the base sequence of SEQ ID NO: 1. A probe can be used.

  Hybridization can be detected by a method widely used in this technical field. For example, the probe can be labeled with a fluorescent substance, radionuclide, digoxigenin (DIG) or the like, and fluorescence, radiation, binding to an anti-DIG antibody, or the like can be detected.

  The determination by the sequence method can be performed as follows, for example. First, a DNA fragment containing the MWG655A sequence is amplified by PCR using the test barley genomic DNA as a template. Then, direct sequencing is performed using the amplified DNA fragment (PCR product) as a template. Alternatively, any vector incorporating the PCR product may be used to transform E. coli, yeast, etc., a transformant retaining the PCR product may be selected, and sequencing may be performed using the vector retained by the transformant as a template. Good. Alternatively, direct sequencing may be performed by PCR using a vector retained by the selected transformant as a template.

  The determination by the CAPS method can be performed as follows, for example. First, PCR is performed using a primer set capable of amplifying the MWG655A sequence, using the test barley genomic DNA as a template, and the resulting amplified DNA fragment (PCR product) is digested with the restriction enzyme HinfI. Then, the size and / or number of restriction enzyme-treated DNA fragments are analyzed to determine whether there is a DNA fragment cleaved by HinfI.

  In the method for selecting barley of the present invention, it is determined whether or not the test barley has a recognition sequence for the restriction enzyme HinfI in the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1 in terms of ease of work and efficiency. And preferably based on the CAPS method.

  That is, the barley selection method of the present invention is, in a preferred embodiment, a method for selecting barley with a high malt extract, and a primer set capable of amplifying the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1. And a PCR step using the genomic DNA of the test barley as a template to obtain an amplified DNA fragment, and a digestion step of digesting the amplified DNA fragment with the restriction enzyme HinfI. This is a method for selecting the test barley as barley having a high malt extract when a DNA fragment cleaved by a restriction enzyme is present in a restriction enzyme-treated product (restriction enzyme-treated DNA fragment). After the digestion step, an analysis step for analyzing the size and / or number of restriction enzyme-treated DNA fragments may be further performed.

  The primer set only needs to be capable of amplifying the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1. Such a primer set can be easily designed by those skilled in the art based on the nucleotide sequence of SEQ ID NO: 1.

  The primer set is composed of a forward primer and a reverse primer, at least one of which is a base sequence of SEQ ID NO: 1 in the result of multiple alignment of the base sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Those that anneal to the polymorphic site in the nucleotide sequence of SEQ ID NO: 1 that is different from the sequence are preferred.

  In the present invention, “multiple alignment” means that a plurality of base sequences can be compared with each other, so that a space (gap) is appropriately inserted so that the number of matching bases is as large as possible. This refers to aligning base sequences. Multiple alignment can be performed using a known multiple alignment creation program (Clustal W, Clustal X, etc.).

  FIG. 1 shows the result of multiple alignment of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The base sequence of SEQ ID NO: 1 (indicated as “SEQ-1” in FIG. 1) is a sequence in the MWG655A locus. The base sequence of SEQ ID NO: 2 (indicated as “SEQ-2” in FIG. 1) is a sequence in the MWG655B locus. The base sequence of SEQ ID NO: 3 (indicated as “SEQ-3” in FIG. 1) is a sequence in the MWG655C locus. In FIG. 1, the base portion marked with “*” indicates a base portion where the above three base sequences match, and “−” indicates a gap. In addition, the underlined portion in the sequence of FIG. 1 is a portion having the same base sequence as the base sequence of the primers (MWG-A3, MWG-C2) used in the examples or a complementary sequence thereof. The base sequences of SEQ ID NOs: 1 to 3 were obtained by sequencing using genomic DNA extracted from barley of the Mikamo Golden / Harlington doubling haploid (MH-DH) strain as a template.

  As is clear from FIG. 1, the base sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 have very high base sequence homology. Further, in the barley chromosome, orthologs having very high homology between the sequence in the MWG655A locus and the base sequence are present in each of the MWG655B, MWG655C and MWG655e loci. These orthologs can be amplified non-specifically together with the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1 depending on the primer sequence used for PCR.

  If at least one of the forward primer and the reverse primer anneals to the polymorphic site, a mismatch occurs between at least one of the forward primer and the reverse primer and the ortholog in the MWG655B and MWG655C loci. For this reason, it is suppressed that the nucleotide sequences in the MWG655B and MWG655C gene loci are non-specifically amplified by PCR, and the subsequent genotyping by restriction enzyme treatment can be performed more easily.

  In the present invention, the length of the primer (forward primer or reverse primer) is not particularly limited, but can be, for example, 7 bases or more, preferably 10 bases or more, and 15 bases or more. It is more preferable. For example, it can be 50 bases or less, preferably 40 bases or less, and more preferably 30 bases or more.

  As a forward primer, for example, a primer containing the base sequence of SEQ ID NO: 4 is preferable, and a primer consisting of the base sequence of SEQ ID NO: 4 is more preferable. Moreover, as a reverse primer, what contains the base sequence of sequence number 5, for example is preferable, and what consists of a base sequence of sequence number 5 is more preferable.

  The genomic DNA of the test barley can be obtained, for example, by performing extraction by a method commonly used for extracting genomic DNA from plant tissues (CTAB method or the like). Commercially available genomic DNA extraction kits can also be suitably used. Genomic DNA may be extracted from any part of the barley leaves, stems, roots, seeds, etc., but leaves are preferred when the barley selection method of the present invention is carried out at the initial stage of breeding.

  As the thermostable polymerase used for PCR, for example, commercially available thermostable polymerases (Premix Taq, Ex Taq version 2.0 (TAKARA Bio), etc.) can be appropriately selected and used.

  The temperature during annealing is preferably 60.0 to 65.0 ° C, and can be 62.5 ° C, for example. By setting the annealing temperature within this range, amplification of the non-target region can be suppressed more efficiently.

  The digestion of the PCR product with the restriction enzyme HinfI can be performed at an optimal temperature in a buffer solution optimal for the restriction enzyme HinfI. For example, it can be carried out at 37 ° C. in a buffer solution (20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Magnesium acetate, 1 mM Dithiothreitol, pH 7.9).

  Analysis of the size and / or number of restriction enzyme-treated DNA fragments can be performed, for example, by agarose gel electrophoresis. Alternatively, the fragmentation can be performed by size fractionation by a HPLC method using a known appropriate column.

  According to the knowledge of the present inventors, barley has the same genotype as the barley Mikamo golden species depending on whether or not the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1 has a recognition sequence for the restriction enzyme HinfI. Hereinafter referred to as “Mi type” or “Mi type genotype”) and those having the same genotype as the barley Harrington species (hereinafter referred to as “Hr type” or “Hr type genotype”). Mi-type barley has higher malt extract than Hr-type barley.

  Therefore, DNA markers for selecting barley with a high malt extract are not limited to those in the MWG655A sequence corresponding to the nucleotide sequence of SEQ ID NO: 1, but are classified into Mi type and Hr type in the MWG655A locus. Any DNA marker that exhibits a possible polymorphism can be used. That is, barley having a high malt extract can also be selected by identifying the genotype of the test barley based on such a DNA marker and selecting the barley identified as the Mi type.

[Barley of the progeny of the cross]
The progeny line of barley of the present invention is a progeny line of barley that can be obtained by crossing barley selected by the barley selection method. The barley selected by the above barley selection method has the MWG655A locus genotype related to the malt extract, which is Mi type. It becomes.

[Mort production method]
The malt production method of the present invention is a method including a malting step of obtaining malt using barley selected by the barley selection method or barley of a progeny line thereof. Since malting is performed using barley selected as barley having a high malt extract or barley of a progeny of the cross, the malt obtained by this method has a high malt extract. Malting can be performed by a known method. For example, after malting until the degree of soaking reaches 40% to 45%, germination is performed at 10 to 20 ° C. for 3 to 6 days, and malt is obtained by drying. be able to.

[Method for producing malt fermented beverage]
The method for producing a malt fermented beverage of the present invention includes a preparation step and a fermentation step, and in the preparation step, barley selected by the above-mentioned barley selection method, barley of the above-mentioned mating progeny line or the above-mentioned malt is used as a raw material. .

  The preparation step is a step of saccharifying malt to obtain wort. More specifically, the process of mixing the raw material containing malt and barley and the water for charging, heating the resulting mixture to saccharify the malt and barley, and collecting wort from the saccharified malt and barley It is.

  The malt used in the preparation step is preferably one obtained by germinating barley with moisture and air in the malting step, drying and removing the young roots. Malt is a major source of starch as a raw material for saccharification as well as an enzyme source necessary for wort production. In order to give the flavor and pigment peculiar to a malt fermented drink, it is preferable to use what dried the germinated malt for wort manufacture. In addition to malt, auxiliary materials such as barley, corn starch, corn grits, rice, and sugars may be added as part of the raw material. Moreover, you may use the malt extract prepared from common barley, a barley decomposition product, processed barley, etc. as a part of raw material.

  What is necessary is just to select suitable water for the water for preparation according to the malt fermented drink to manufacture. Further, saccharification may be performed under general conditions. After filtering the malt saccharified solution thus obtained, raw materials (hops, herbs, etc.) that can impart aroma, bitterness, etc. are added and boiled, and cooled to obtain cold wort.

  A fermentation process is a process of adding yeast to the cold wort obtained at the preparation process, making it ferment, and obtaining a malt fermented drink. Examples of the yeast used here include Saccharomyces patrianus, Saccharomyces cerevisiae, Saccharomyces ubalum and the like.

  The malt fermented drink should just be a drink manufactured by fermentation using barley or malt as a part of raw materials. Examples of such beverages include beer and happoshu. In addition, so-called non-alcohol beer and non-alcohol sparkling liquor are also produced as malt fermented drinks because they are produced by the same manufacturing method as beer and the like.

  Hereinafter, the present invention will be specifically described based on examples. In the following examples, “MWG655A sequence”, “MWG655B sequence”, “MWG655C sequence” and “MWG655e sequence” mean base sequences in the MWG655A, B, C and e loci, respectively. .

[QTL analysis of malt extract]
(Barley sample)
Mikamo Golden / Harlington doubled haploid (MH-DH) 95 barley (produced in 1998 in Tochigi, 2002 in Tochigi, and in 2006 in Canada) were used as samples.

(Measurement of malt extract)
The malt was obtained by making the barley seeds of the above MH-DH line by small malting. After measuring the dry weight of the obtained malt, the congress wort was prepared. The specific gravity of Congress wort was measured using a vibratory densitometer (Density Meter DMA4500M: manufactured by Anton Paar) and published in the literature [European Brewery Convention, Analytical (1987) European Brewery Convent. The malt extract was measured by the European Brewery Convention (EBC) standard method described in Brauerei und Getränke Rundschau, Zurich].

(QTL analysis)
Mikamo Golden and Harrington DNAs extracted by the CTAB method were subjected to single nucleotide polymorphism (SNPs) analysis using Illumina GoldenGate BeadArray. About the SNPs from which the polymorphism was obtained, polymorphism investigation was carried out in the MH-DH line. Furthermore, polymorphism analysis of MH-DH was also performed on RFLP, hordein, SSR, and EST markers. A linkage map was created using 550 polymorphism data of the MH-DH line. QTL analysis was performed using this map. QTL analysis was performed using JoinMap v3.0 (http://www.kyazma.nl/index.php/mc.JoinMap/).

(result)
As a result of QTL analysis of malt extract by three kinds of MH-DH lines (from 1998 and 2002, Tochigi, 2006 from Canada) having different origins and production years, chromosome 2H (QTL peak: 33. The malt extract QTL was detected at 2 cM, contribution ratio: 14.7 to 22.7%. When the effect of this QTL was verified, it was found that the genotype of the MWG655A locus can be classified into Mi type and Hr type. Furthermore, the malt extract was significantly higher in the MWG655A gene locus at the Mi type than at the Hr type in any production region and year (FIG. 2). In FIG. 2, each data is shown as mean ± standard deviation. Further, “**” attached to the Mi type data indicates that the data is data having a significant difference (risk rate: 1%) as compared with the Hr type. The presence or absence of a significant difference was determined using a t-test. In FIG. 2, the reason why the malt extract of 2006 Canadian barley is lower than that of Tochigi is considered to be because the crude protein content is higher than that of Tochigi.

  From the above, it was suggested that if the genotype of the MWG655A locus is the Mi type, the malt extract becomes higher. Moreover, since it is based on the analysis using the MH-DH system barley from which a production year and a production area differ, it was thought that it became a highly versatile DNA marker.

[Construction of CAPS marker]
The MWG655A marker is known as the RFLP marker. Identification of the genotype of the MWG655A locus by RFLP is carried out using the MWG655 clone as a probe, and four different markers A, B, C and e are identified by the four band patterns detected. The MWG655 locus has at least four similar base sequences, A, B, C, and e, but each base sequence has not been known so far.

  In order to analyze the MWG655A sequence, amplification of the MWG655A sequence by PCR was attempted. For this purpose, first, the base sequence of the MWG655 clone as an RFLP probe was analyzed, and primers were designed at the 5 ′ and 3 ′ ends of the MWG655 clone based on the obtained base sequence (MWG-3, MWG-4). . Using this primer set (MWG-3, MWG-4), PCR was performed using Mikamo Golden species and Harrington species genomic DNA as templates. When the amplified DNA fragments were sequenced and their base sequences were compared, polymorphisms were detected at several locations.

  Among the detected polymorphisms, a single nucleotide polymorphism containing a restriction enzyme HinfI recognition sequence was used as a temporary CAPS marker to determine the genotype of MH-DH95 strain barley. separated. However, since the MH-DH line is a doubled haploid and is genetically fixed, if the amplified DNA fragment is derived from any one of the MWG655A, B, C, and e loci, the heterozygote The type should not appear. Therefore, it was considered that two or more kinds of MWG655A, B, C and e sequences were mixed in the DNA fragment amplified with this primer set (MW-3, MW-4).

  Then, the comparative analysis with the pattern by this provisional CAPS marker and RFLP pattern was performed about the MH-DH type | system | group barley which shows Mi type | mold, Hr type | mold, and a hetero type | mold. As a result, barley showing the same genotype in the RFLP pattern for MWG655B and C shows Mi type or Hr type in this provisional CAPS marker, and barley showing a different genotype in the RFLP pattern shows heterotype in this provisional CAPS marker. Indicated. That is, it was suggested that at least the MWG655B and C sequences were mixed in the DNA fragment amplified with this primer set (MW-3, MW-4).

  Furthermore, the base sequence of the heterozygous DNA fragment detected with this provisional CAPS marker was analyzed using the RFLP results for the MH-DH strain barley that showed heterozygote, and MWG655B and C sequences were obtained (SEQ ID NO: 2, 3). Based on the polymorphism existing between these base sequences and the base sequence of the MWG655 clone, primers MWG-C2 (a primer that does not amplify MWG655B) and MWG-7 (a region in the MWG655 clone containing the polymorphic site) Have the same base sequence). PCR was performed using MWG-7 and MWG-C2, and the base sequence of the obtained DNA fragment was analyzed (SEQ ID NO: 1).

  By comparing the nucleotide sequences of SEQ ID NOs: 1 to 3, a polymorphism was detected between the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequences of SEQ ID NOS: 2 and 3 (FIG. 1). Primers were designed so as to include a polymorphic site in the base sequence of SEQ ID NO: 1 (MWG-A3), and PCR was performed using MWG-A3 and MWG-C2. The obtained amplified DNA fragment was digested with each of 45 major restriction enzymes and screened for restriction enzymes. As a result, it was found that when digested with HinfI, the genotype of MH-DH95 barley was consistent with the RFLP results for the MWG655A sequence.

  As shown in FIG. 3, when agarose gel electrophoresis was performed on the HinfI-treated product, a band was detected at about 240 bp in the Mi type, but this band was not detected in the Hr type. In addition, a band was detected at about 270 bp for both Mi type and Hr type, but this band was considered not to be the MWG655A sequence but to the MWG655e sequence having a base sequence similar to the MWG655A sequence (in the MH-DH strain, the MWG655e gene locus). Does not exist). In FIG. 3, the band indicated by the arrow is a band of about 240 bp detected only by the Mi type. The presence or absence of this band makes it possible to identify the Mi type (with band) or Hr type (without band).

  Based on the above, the barley genotype can be identified based on whether the base sequence of SEQ ID NO: 1 is the MWG655A sequence and whether or not the recognition sequence of HinfI is present in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1 (present) In this case, it was confirmed that it was Mi type, and if not, Hr type).

[Barley selection by CAPS marker]
(Barley sample)
38 major beer varieties cultivated in Sapporo Beer's Kizaki field (2000: 15, 2004: 30, 2005: 29, 2006: 27, 2009: 26) did.

(Measurement of malt extract)
The measurement of the malt extract was performed in the same manner as described above.

(CAPS method)
PCR was performed under the following conditions using the genomic DNA of each variety as a template and the following primers. The obtained PCR product was digested with HinfI and then subjected to agarose gel electrophoresis, and the genotype was identified by the band position (DNA fragment length).
MWG-A3 (forward primer): 5′-TGTAGTCAGTCCAGGGTGTATTCCAC-3 ′ (SEQ ID NO: 4)
MWG-C2 (reverse primer): 5′-CGGGTTGAACTATGTCTATTC-3 ′ (SEQ ID NO: 5)
PCR conditions: 94 ° C., 1 minute denaturation, 62.5 ° C., 1 minute annealing, 72 ° C., 5 minutes extension reaction as one cycle, this is repeated 35 cycles, followed by 70 ° C., 5 minute extension reaction Do.

(result)
Of the 38 varieties examined, 5 were genotypes showing Mi type and 33 were showing Hr type. The breakdown by production year is as follows.
2000: Mi type 3 / Hr type 12 type 2004: Mi type 4 type / Hr type 26 type 2005: Mi type 3 / Hr type 26 2006 2006: Mi type 3 / Hr type 24 : Mi type 3 types / Hr type 23 types

  When the average value of the malt extract for each genotype was compared, the malt extract was significantly higher in the Mi type barley than in the Hr type in any production year (FIG. 4). In FIG. 4, each data is shown as mean ± standard deviation. In addition, “*” and “**” attached to Mi type data indicate that the data is significantly different from the Hr type (risk rate: 5% and 1%, respectively). Show. The presence or absence of a significant difference was determined using a t-test.

  From the above examples, it was confirmed that barley extract having a recognition sequence of restriction enzyme HinfI in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1 is higher in malt extract than other barley. Further, according to the method for selecting barley of the present invention, it was confirmed that barley having a high malt extract can be selected reliably without being affected by the year of production, the place of production, environmental factors and the like.

SEQ ID NO: 4; forward primer MWG-A3
SEQ ID NO: 5: reverse primer MWG-C2

Claims (5)

A method for selecting barley with a high malt extract,
A method of selecting test barley as barley having a high malt extract when the test barley has a recognition sequence of restriction enzyme HinfI in the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1. A method for selecting barley with a high malt extract,
An amplification step of obtaining an amplified DNA fragment by performing a polymerase chain reaction using a genomic DNA of a test barley as a template using a primer set capable of amplifying the MWG655A sequence corresponding to the base sequence of SEQ ID NO: 1, and an amplified DNA fragment A digestion step of digesting with the restriction enzyme HinfI,
A method of selecting the test barley as barley having a high malt extract when a DNA fragment cleaved by the restriction enzyme is present in the restriction enzyme-treated product obtained in the digestion step. The primer set consists of a forward primer and a reverse primer,
At least one of the forward primer and the reverse primer is
In the multiple alignment result of the base sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, the base sequence of SEQ ID NO: 1 is annealed to a polymorphic site in the base sequence of SEQ ID NO: 1 which is different from any other base sequence. The method of claim 2, wherein:   The method according to claim 3, wherein the forward primer includes the first to eleventh base sequences of the base sequence of SEQ ID NO: 1.   The method according to any one of claims 2 to 4, wherein the primer set comprises a forward primer composed of the base sequence of SEQ ID NO: 4 and a reverse primer composed of the base sequence of SEQ ID NO: 5.